4. Material and Methods
4.6 Biochemical Methods
121 supernatant was transferred to a new reaction tube. RNA was then obtained by EtOH precipitation (see section 4.5.2).
4.6 Biochemical Methods
122 (HiLoad 16/600 Superdex 200 pg or Superose 6 Increase 10/300 GL) connected to an FPLC. Separation was performed with a total volume of 120 ml HEPES buffer (50 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7). The absorbance at 280 nm was continuously measured by a built-in UV detector. Proteins in the eluted samples were identified by SDS-PAGE, while RNA was detected by Urea-PAGE. The total amount of purified protein was determined using Bradford assay.
4.6.4 Anion-exchange chromatography
Anion-exchange chromatography was performed for small RNA wrapping complexes formed on repeat-tagged sfgfp RNA produced T7 RNAP following the protocol of Jahn et al. (Jahn et al., 1991). The concentrated sample was loaded on a MonoQ column and the flow-through was collected. The bound sample was gradually eluted in a linear gradient over 20 column volumes and an increasing concentration of NaCl (50 mM Tris/HCl, 300-1000 mM NaCl, 1 mM DTT, 10 % glycerin, pH 7). To remove the remaining bound protein, the column was further washed with 5 CV of wash buffer (50 mM Tris/HCl, 300 mM NaCl, 1 mM DTT, 10 % glycerin, pH 7).
4.6.5 Protein quantification by Bradford
The amount of purified protein was measured by Bradford assay (Bradford, 1976). 200 µl Bradford reagent was added to a dilution of the protein sample and the mix was incubated for 15 min . Afterwards, the OD595nm was measured and the amount of protein was calculated by a fresh calibration curve with BSA.
4.6.6 Production and purification of recombinant proteins
4.6.6.1 Purification of recombinant I-Fv Cascade and truncated variants
For production and purification of recombinant I-Fv Cascade, cas genes and crRNA were co-purified. The cas genes cas7fv, cas5fv, cas6f were provided in the vector pRSFDuet-1 which allows for the simultaneous production of all three proteins with Cas5fv fused to an N-terminal His-tag. This plasmid was co-transformed into E. coli BL21 (DE3) pLys with a second pUC19 vector containing the repeat-spacer4-repeat sequence of the single S. putrefaciens CN-32 CRISPR array downstream of a T7 RNA polymerase promoter. Cultures were grown and harvested as described (see section 4.2). Cell pellets were lysed and proteins first purified via Ni-NTA purification in a buffer containing 50 mM Tris/HCl, 20-500 mM imidazole, 300 mM NaCl, 10 mM MgCl2, 1 mM DTT, 10 % glycerine, pH 7. Samples containing protein as detected by UV were then pooled, concentrated to 2 ml and subjected to size-exclusion
123 chromatography (HiLoad 16/600 Superdex 200 pg) in a buffer containing 50 mM HEPES-NaOH pH 7.0, 150 mM NaCl.
Truncated Cascade, missing the AH domain of Cas5fv or missing the wrist loops from Cas7fv as well as Cascade with the sfGFP-Cas7fv fusion purified were by co-production of the respective genes from pRSFDuet with a crRNA from pUC19 in the same fashion as for wt-Cascade.
4.6.6.2 Purification of recombinant Cas3fv and Cas1-Cas2/3fv complex
The target nuclease Cas2/3fv was purified as a fusion construct with an N-terminal His-tag and a C-terminal MBP-Tag using the vector pETM-43. Standalone Cas3 with the Cas2 portion removed was provided on a pet24(+) vector by Dr. Patrick Pausch. Cas3 was produced and purified via Ni-NTA chromatography and size exclusion chromatography (Superose 6 Increase 10/300 GL) in a buffer containing 20 mM HEPES/KOH pH 7.5, 750 mM NaCl, 10 mM MgCl2 and 2% Glycerin at 4 °C. The Cas1-Cas2/3 complex was purified by co-expression of cas1 and cas2/3fv from pRSFDuet-1 with an N-terminal Strep-tag fused on Cas1. The final size-exclusion chromatography was performed with HiLoad 16/600 Superdex 200 pg.
4.6.6.3 Purification of filaments and RNA wrapping complexes
For directed RNA wrapping a repeat-tagged sequence, either sfgfp, lacZ-α or a non-coding sequence (from the backbone of the pRSFDuet vector) were transcribed from a pBAD vector for arabinose induction or pETDuet-1 for induction with IPTG. Cas genes were produced from pRSFDuet or pCsy_complex (addgene). In a later experiment, cas genes were split on different vectors. In this setup, cas7fv was expressed from pRSFDuet (either with a copy in the first MCS or with a copy in both MCS of the plasmid) and cas5fv and cas6f were expressed from pACYCDuet. Repeat-tagged target transcripts were expressed from pBAD as before.
Formed complexes were purified via Ni-NTA chromatography in a standard purification buffer (50 mM Tris/HCl pH 7.0, 300 mM NaCl, 20-500 mM imidazole, 10 mM MgCl2, 10% glycerol, 1 mM DTT) as for Cascade purification. For further clean-up and TEM analysis, size-exclusion chromatography was performed in the same fashion as for I-Fv Cascade and samples from the fractions of the void volume were taken for analyses. For double affinity purification, a C-terminal Strep-tag was fused to Cas6f in addition to the N-terminal His-tag of Cas5fv on the pRSFDuet plasmid and Cas proteins were produced and purified via Strep-tag affinity chromatography.
To remove rRNA, Purification was later repeated by Ni-NTA under the same conditions as before but without MgCl2 in the wash buffer (50 mM Tris/HCl, 300 mM NaCl, 1 mM DTT, 10 % glycerin, pH 7).
124 Samples were concentrated and loaded on an analytic size-exclusion (Superose 6 Increase 10/300 GL) in the standard SEC buffer of I-Fv Cascade.
For purification of small RNA wrapping complexes, repeat-tagged sfgfp was produced from pETDuet with T7 RNAP and co-purified by Ni-NTA with Cas proteins via His-tagged Cas5fv. Samples were concentrated to a final volume of 500 µl and subjected to Anion-exchange chromatography. Fractions were collected in 700 µl and analyzed by SDS- and Urea-PAGE. The flow-through was pooled and subjected to size-exclusion chromatography with a small semi-preparative and analytic column (Superose 6 Increase 10/300 GL). Samples were eluted in standard SEC buffer.
4.6.6.4 Purification of SUMO-Cas7fv
His-SUMO-Cas7fv was purified with the standard purification buffer for Cascade but an extra wash step with a high salt buffer (50 mM KCl, 1 M NaCl, 10 mM MgSO4, 2 mM ATP) was applied for 20 column volumes while protein was bound to the column. Protein was the n eluted with standard Cascade elution buffer containing imidazole and eluted protein was concentrated before being loaded on size -exclusion chromatography at 4 °C to separate monomeric SUMO-Cas7fv from aggregated protein.
4.6.7 In vitro RNA wrapping
500 µl RNA-free SUMO-Cas7fv or Cas5fv-Cas7fv dimer (~ 1 mg) were mixed with either in vitro transcribed sfgfp RNA (~ 5 µg) or small RNA extracted from pseudomonas oleovorans in our laboratory (3.5 µg, heated up at 95 °C and cooled down on ice before added to protein) and incubated for 1 h at RT followed by incubation overnight at 4 °C (with the addition of SUMO protease (1:500) in case of Cas7fv-SUMO). The following day, precipitated protein was removed by centrifugation and the supernatant was loaded on size-exclusion chromatography (Superose 6 Increase 10/300 GL) to separate potential complexes from monomeric protein.
Crystallization of Cas7fv was performed in hanging-drop format or in microtubes following a protocol established by Dr. Patrick Pausch. Freshly purified I-Fv Cascade (concentrated to 54 mg/l) was mixed in various ratios (2:1, 1:1, 1:2) with screening solution (0.2 M MgCl, 0.1 M Tris pH 7.2, 2 M NaCl and 3 % fructose) and incubated at 18 °C until crystals were formed.
4.6.8 RNA protection assays
Ni-NTA purified RNA wrapping complexes formed with on the lacZ-Repeat construct were incubated at 23 °C, 200 rpm and 10 U of RNase I for varying time points of up to 7 days. Following this, RNA was extracted and separated on Urea-PAGE. 1 µg of directly extracted RNA was further incubated under the
125 same conditions to ensure the activity of RNase I and then separated on Urea-PAGE. Gels were either stained with Sybr-Gold for nucleic acid detection or used for Northern Blot analysis. The 50 bp DNA ladder (NEB) was used for orientation.
4.6.9 Electrophoretic mobility shift assays (EMSA)
The recombinant Cascade complex was tested for its ability to bind target DNA constructs in electrophoretic mobility shift assays (EMSAs). Utilized target oligonucleotides contained a sequence complementary to the spacer sequence in the crRNA (spacer4) with a correct PAM (GG) or a wrong PAM (TT) at the 3′-end adjacent to the spacer. A non-complementary spacer sequence (spacer1) was used as a control. For dsDNA constructs, the non-target strand was labeled to ensure that a shift in EMSA is due to fully hybridized construct (see section 4.4.9). Target and non-target strand were hybridized by incubation for 10 min at 95 °C and cooling down to RT over 2 hours. A total of 20 nM (∼20,000 cpm) of labeled substrate was incubated with varying concentrations of Cascade (0–60 nM) for 30 min at 30 °C in 50 mM HEPES-NaOH pH 7.0, 150 mM NaCl and 1 mM DTT. Samples were mixed with Gel Pilot loading dye (Qiagen) and separated via non-denaturing TBE-PAGE (6% polyacrylamide, 1x TBE).
4.6.10 Nuclease assays
Nuclease assays were performed to study in vitro interference of Cas3fv and the Cas1-Cas2/3fv complex.
To study activity of Cas3fv on ssDNA, a consistent amount of protein (500 nM) was incubated with 1 µg ssDNA substrate (M13mp18 ssDNA, NEB) over increasing time points at 30 °C in a buffer containing 20 mM HEPES pH 7, 100 mM NaCl, 5 mM MgCl2, 5 mM MnCl2 and 1 mM ATP. To compare ssDNA activity of Cas3fv and Cas1-Cas2/3fv, increasing amounts of protein (0, 50, 100, 200 and 500 nM) were incubated with 1 µg ssDNA substrate with the same buffer. 10 mM EDTA was added to stop the reaction in control samples. Remaining ssDNA substrate was separated and visualized by gel electrophoresis in 1x TBE (see section 4.4.4.1).
To study the activity on dsDNA substrates containing a small 10 nt “bubble” opening and Cascade -bound R-loop substrates (created by incubation with Cascade as in 4.6.9), radioactively labeled substrates were used. In these assays, ~ 20,000 cpm of substrate was incubated without protein or with 500 nM Cas3fv or Cas1-2/3fv for 2 h at 30 °C. Samples were separated via denaturing PAGE (see section 4.5.4.2) and gels were visualized via phosphoimaging. Radioactively labeled Low Molecular Weight Marker (Affymetrix) was used for size determination.
126 4.6.11 Crystallization and 3D structure analysis of I-Fv Cascade
Crystallization of I-Fv Cascade and solving of the 3D structure was performed by Dr. Patrick Pausch in the laboratory of Prof. Dr. Bange of the Philipps-University Marburg (Pausch et al., 2017). Purified Cascade was concentrated to an absorbance at 280 nm of 35 AU (NanoDrop Lite Spectrophotometer) and crystals were formed by hanging drop vapor-diffusion at 20 °C. Crystallization of the Se-Met labeled Cascade was conducted in a two-step protocol, based on the initial formation of seed-crystals derived from native Cascade complexes. Seed crystals were generated by mixing equal volumes (1 μl) of Cascade sample and crystallization buffer (16% w/v PEG6000, 0.1 M Tris pH 8.0 and 20 mM 5-amino-2,4,6-triiodoisophthalic acid). Sword-shaped seed crystals grew overnight and were subsequently used for streak seeding with a cat whisker into crystallization drops containing Se -Met labeled Cascade. Crystals grew within days in drops containing 1 μl of Se-Met labeled Cascade sample and 1 μl crystallization buffer (18% w/v PEG6000, 0.1 M Tris pH 7.6 and 20 mM 5-amino-2,4,6-triiodoisophthalic acid). Crystals were transferred into crystallization buffer containing 20% v/v glycerol as cryo-protectant, subsequently flash frozen and stored in liquid nitrogen. R-loop/Cascade samples were concentrated to an absorbance at 280 nm of 30 AU (NanoDrop Lite Spectrophotometer) and crystals were formed by hanging drop vapor-diffusion at 20 °C. Needle shaped crystals grew within days in drops containing 1 μl of R-loop/Cascade and 1 μl crystallization buffer (22.5% w/v PEG4000, 15% v/v glycerol, 153 mM ammonium acetate and 85 mM sodium citrate pH 5.6). R-loop/Cascade crystals were flash-frozen and stored in liquid nitrogen.
Diffraction data was collected at beamline ID29 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Data was processed with the XDS program package for data reduction (Kabsch, 2010), Crank2 for experimental phasing of Se-Met labeled Cascade (CCP4 package, (Winn et al., 2011), coot (Emsley & Cowtan, 2004) in combination with Refmac5 (CCP4 package) and phenix.refine (PHENIX package) for iterative model building and refinement (Adams et al., 2010). The R-loop/Cascade dataset was solved by molecular replacement using the Cascade structure via the CCP4 implemented program Phaser (McCoy et al., 2007). Figures were prepared in Pymol.
4.6.12 Electron Microscopy
Transmission Electron Microscopy was performed by Dr. Thomas Heimerl of the Philipps -University Marburg to visualize filament structures and small RNA wrapping complexes. 2D Class averaging was performed to obtain a 3D model by combining a total of 30 montages.
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